Project summary Post-translational modifications (PTMs) remarkably expand both the chemical as well as the functional space accessible to human proteins. A key mechanism through which the PTM of a protein leads to new functional outcomes involves the participation of the modified protein in unique protein-protein interactions (PPIs) that are inaccessible to its unmodified counterpart. Such PPIs are often driven by designated ?reader? proteins that directly bind the modified amino acid residue. Additionally, PTMs can also trigger new PPIs indirectly by altering the conformational state or the subcellular localization of the protein. Over the last two decades, advances in proteomic technologies and mass-spectrometry have dramatically expanded the catalog of known PTMs within the human proteome. However, the physiological roles for the majority of these PTMs remain elusive. Identifying functionally important PPIs associated with an orphan PTM remains a particularly daunting challenge, because: 1) generating proteins homogeneously labeled with a PTM at the desired site is difficult, as the mechanisms of their installation are often unclear or hard to reconstitute, and 2) the weak and transient nature of such PPIs make them challenging to capture using traditional biochemical strategies. We propose an innovative and general solution to overcome these challenges by developing a technology that enables co-translational, site-specific incorporation of both the modified amino acid as well as a photoaffinity probe into any two sites of any protein. The ability to generate such ?protein- probes? homogeneously modified with the PTM at the desired site and further equipped with a photo-crosslinker group, the position of which can be systematically altered to optimally capture interaction partners, will provide a general strategy to systematically identify novel PPIs triggered by a PTM in the human proteome. We will initially focus on a suit of lysine-PTMs associated with our histones that regulate transcription and chromatin dynamics, but our technology will be broadly applicable to many other PTMs that have been genetically encoded. Furthermore, to achieve the aims described herein, we will develop two core technologies that will have broad applications far beyond the scope of this proposal: 1) Optimized platforms for incorporating two distinct non-canonical amino acids (ncAAs) into proteins expressed in mammalian cells. This would be also useful for additional applications, such as site-specific installation of two small optical probes to monitor protein dynamics. 2) Development of a novel mammalian cell-based directed evolution platform to create improved ncAA-specific aminoacyl-tRNA synthetase (aaRS)/tRNA pairs that efficiently interfaces with the mammalian translation system. Such a directed evolution platform is not available today and, in addition to significantly expanding the scope of the ncAA-mutagenesis technology, it can be readily adopted to evolve numerous other biological functions in live mammalian cells, which will have a broad and deep impact on biomedical research.